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First published online May 9, 2008
doi: 10.1242/10.1242/dev.017160

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The multidomain protein Brpf1 binds histones and is required for Hox gene expression and segmental identity

Kathrin Laue^1,*, Sylvain Daujat^2,*, Justin Gage Crump³, Nikki Plaster^1,, Henry H. Roehl⁴ Tübingen 2000 Screen Consortium, Charles B. Kimmel⁵, Robert Schneider^2,, Matthias Hammerschmidt^1,6,

¹ Georges-Koehler-Laboratory and
² Hans-Spemann-Laboratory, Max-Planck-Institute of Immunobiology, Stuebeweg 51, D-79108 Freiburg, Germany.
³ Center for Stem Cell and Regenerative Medicine, USC Keck School of Medicine, Los Angeles, CA 90033, USA.
⁴ Centre of Developmental and Biomedical Genetics, University of Sheffield, Sheffield S10 2TN, UK.
⁵ Institute of Neuroscience, 1254 University of Oregon, Eugene, OR, USA.
⁶ Institute for Developmental Biology, University of Cologne, D-50923 Cologne, Germany.

View larger version (90K): [in this window] [in a new window]   Fig. 1. Zebrafish brpf1 mutants display anterior shifts in pharyngeal arch identities. Genotypes of fish are indicated in upper right corners (WT, wild type; -/-, homozygous brpf1 mutant; MO, brpf1 morphant), stages in lower right corners. (A,B) Lateral views of live larvae. (C-L) Cartilaginous elements of visceral skeleton stained with Alcian Blue (AB). (C-I) Ventral views; neurocranium has been removed. Numbers of pharyngeal arches are indicated (1-7). Arrowheads (D,E,G) point to absent basihyal (bh) of mutant arch 2. In addition, arches 3 and 4 of the brpf1 mutant lack hypobranchials (hb) (G, asterisks), intermediate elements that (in wild-type larvae) are characteristic for arches 3-7, but absent in arches 1 and 2 (C,F). Furthermore, the distal ends of the mutant ceratobranchials (cb) (I; 3,4) have acquired the shape and organization of the ceratohyal (ch) of the second arch of wild-type larvae (H; 2). (J-L) Lateral views of arches 1 and 2. Arrows in K point to joints between ventral and dorsal elements (compare with N). Arrow in L points to fusion between Meckel's cartilage (m) of arch 1 and the transformed ceratohyal (ch) of arch 2, an ultimate sign of segmental identity. Note the variable loss of cartilage dorsal of the foramen (f) (K,L), the reduction of the symplectic extension (sy) of the transformed hyosymplectic (hs) and its fusion with the interhyal (ih) (K), which is an arch 2-specific linker element absent in arch 1 (J), giving the hyosymplectic a spatial organization more similar to that of the palatoquadrate (pq). (M,N) Lateral views of head region ventral to eyes after in situ hybridization for bapx1, a first arch joint marker. Arrow points to ectopic bapx1 expression in arch 2 of the brpf1 mutant. (O,P) Lateral views of head region posterior to eyes after immunostaining of pharyngeal muscles with anti-MF20 antibody. (Q-T) Lateral (Q,R) and ventral (S,T) views of heads after staining of bone matrix with Alizarin Red (AR). Arrows point to absent ossification in ceratohyal (ch, ventral element; R) and hyomandibula (hm, dorsal element; T) of arch 2 in the mutant. In addition, the branchiostegal rays (bsr) and the opercle (op) dermal bones associated with the ventral and dorsal element of arch 2, respectively, are absent (arrowhead in R) or reduced. Furthermore, ceratobranchials (cb) of arches 3-6 display ectopic central ossifications (T), as in the wild-type ceratohyal of arch 2 (S). By contrast, arch 7 appears normal (S,T), with characteristic pharyngeal teeth formation (Van der Heyden et al., 2001). (U) Genetic and physical map of the t20002 allele of zebrafish brpf1. The three brpf1 exons on genomic fragment NA5599 are indicated in red. (V) Schematic of predicted wild-type and t20002, b943 and t25114 mutant Brpf1 proteins, with the C2H2, PHD finger, bromo and PWWP domains in different colors. am, adductor mandibulae; bb, basibranchial; ih, interhyal; lap, levator arcus palatini.

View larger version (95K): [in this window] [in a new window]   Fig. 2. Brpf1 regulates segmental identity by maintaining anterior Hox gene expression. Whole-mount in situ hybridizations with the probes indicated bottom left at the stages indicated bottom right; genotypes and treatment of zebrafish embryos as indicated in upper right corners. (A-L) Lateral views; (M-P) ventral views. (A-D) Hox gene expression in wild type (WT). Hox-expressing hindbrain rhombomeres (r) and arch-forming cranial neural crest (CNC) (2-7) are indicated. sc, spinal cord. (E-H) Absent or reduced Hox gene expression in brpf1 mutants (-/-). Arrow in H indicates the remaining hoxb3a expression in the posterior CNC. (I-L) Partially rescued Hox gene expression in the hindbrain (I, arrow) and the CNC (J-L, arrows) of brpf1 mutants injected with mouse Brpf1 mRNA. (M-P) The bimandibular phenotype of the brpf1 mutant (N) can be overcome by injection of hoxb1a mRNA. (O) hoxb1a-injected wild-type embryo lacking bapx1 expression, indicative for bihyoid phenotype [compare with Hunter and Prince (Hunter and Prince, 2002)]. (P) hoxb1a-injected brpf1 mutant with bihyoid pattern on left side and wild-type pattern on right side. Arch numbers are indicated.

View larger version (86K): [in this window] [in a new window]   Fig. 3. Expression pattern and cell-autonomous function of brpf1 in zebrafish CNC. Staining with reagents indicated at lower right at the stages indicated upper right. Numbers of pharyngeal arches are indicated (1-7). (A-J) Wild-type embryos; (K) brpf1b943 mutant (-/-); (L-O) mutant transplanted with wild-type cells (WT -/-). (A) Dorsal view; (B-E,J-O) lateral views; (F-H) horizontal section; (I) longitudinal section. (A-D) At 26 hpf, brpf1 is co-expressed with the CNC marker dlx2a (A) and with fli1a (D), stained by anti-GFP immunostaining of tg(fli1a:EGFP) embryo (Isogai et al., 2003). brpf1-positive cells between CNC include pharyngeal endoderm [D; compare with Fig. 1A in Crump et al. (Crump et al., 2004)]. (E-I) At 55 hpf, sox9a-positive chondrocytes of cartilage condensates (cc; H) (Yan et al., 2002) and pax9a-positive pharyngeal endodermal cells (pe; G) (Nornes et al., 1996; Okabe and Graham, 2004) lack brpf1 expression, which, however, is strongly expressed in p63 (tp63 - ZFIN)-positive cells (Carney et al., 2007) of the pharyngeal ectoderm (pec; F-H), in the oral ectoderm (oe; I) and in facial ectoderm ventral to arches 1 and 2 (vfe; I) (Crump et al., 2006). (J-O) Analysis of chimeric embryos with rhodamine-dextran (RD)-labeled (N) and tg(fli1a:EGFP)-positive (M) wild-type cells integrated in the CNC of a brpf1 mutant host [for procedure, see Crump et al. (Crump et al., 2006)]. Only wild-type, not adjacent mutant CNC, cells display hoxa2b expression (arrows in L and M; n=3/3). g, gut; op, opercle.

View larger version (74K): [in this window] [in a new window]   Fig. 4. Genetic interaction of Brpf1 and Moz and rescue of the brpf1 mutant phenotype by TSA treatment. (A-H) Synergistic enhancement of phenotypes caused by partial loss of Brpf1 and Moz. hoxa2b (A-D; 35 hpf) and bapx1 (E-H; 52 hpf) in situ hybridizations of zebrafish larvae after single or double injections of low amounts of MOs, as indicated in upper right corners. Lateral views. Arrow in D points to absent hoxa2b expression in CNC. Arrow in H indicates ectopic bapx1 expression domain in arch 2. (I-L) Rescued hoxa2b expression in the brpf1 mutant (-/-) after TSA treatment (compare L with K), whereas expression in treated wild-type siblings remains largely unaltered (compare J with I).

View larger version (59K): [in this window] [in a new window]   Fig. 5. Co-localization and physical interaction of Brpf1 and Moz. (A-D) Brpf1 co-localizes with Moz. Immunofluorescent staining of interphase HEK 293 cells after co-transfection with the indicated versions of GFP-Brpf1 (left panels; green) and FLAG-Moz (middle panels; red), counterstained with DAPI (for DNA; blue); merged images are shown in right-hand panels. Full-length Brpf1 co-localizes with wild-type Moz (A) and with HAT-negative Moz-G675E (B) in a punctate pattern on interphase nuclei. Co-localization is abolished when Brpf1 is N-terminally truncated (C). N-terminal fragment of Brpf1 co-localizes with Moz, but displays a more diffuse distribution (D). (E,F) Schematic structures and co-localization/immunoprecipitation properties of full-length Brpf1, full-length Moz (E), and the various truncations used (F). (G-I) Brpf1 physically associates with Moz. (G) Co-IP of full-length Brpf1 and wild-type or HAT-negative Moz(G657E) from co-transfected cells with anti-FLAG (Moz) antibody (left) or anti-HA (Brpf1) antibody (right), analyzed in western blots (upper panels) with the specified antibodies, or assayed for HAT activity on core histones (lower panels). (H) Co-IP of full-length FLAG-Moz and various GFP-Brpf1 deletion constructs with anti-FLAG or anti-GFP antibodies, followed by analysis of complex formation (upper panel) and control for Brpf1 expression levels (lower panel) via anti-GFP western blotting. (I) Co-IP of full-length Moz or C-terminally truncated MozN and various HA-tagged versions of the N-terminal fragments of Brpf1 using anti-FLAG antibody, analyzed in anti-HA western blots. Lower panel shows input control. Brpf1 aa 1-245 fragments that have histidine or cysteine mutations in the zinc-finger domain can still co-precipitate with Moz (lanes 5, 6), whereas the aa 1-149 fragment with an intact zinc finger cannot (lane 4). Scale bars: 5 µm in B; 2.5 µm in D.

View larger version (72K): [in this window] [in a new window]   Fig. 6. The PWWP domain is required for association of Brpf1 with metaphase chromosomes. (A-G) Immunofluorescent staining of mitotic HEK 293 cells transfected with the indicated GFP-Brpf1 constructs (A-E; green) and FLAG-Moz (F,G; red). (A,F) Spreads of metaphase chromosomes. Right panels of A-E and F,G show merged images with DAPI staining of DNA (blue). Full-length Brpf1 displays punctate distribution along metaphase chromosomes (A), whereas in intact nuclei, localization is concentrated in fewer, but still distinct domains of the DNA (B). Truncated Brpf1 lacking the PWWP domain (C) and a Brpf1 fragment containing the PHD domain and the bromodomain (D) are excluded from mitotic chromosomes, whereas a Brpf1 fragment containing the bromodomain and the PWWP domain co-localizes with DNA (E) in a similar manner to full-length Brpf1 (B). (F,G) In contrast to Brpf1 (A,B), no chromatin association is apparent for Moz in metaphase chromosome spreads (F) and in intact mitotic nuclei (G). (H) Schematic structures and chromosome-targeting properties of the full-length and truncated versions of Brpf1. (I-L) Immunofluorescent staining of mitotic HEK 293 cells, revealing that full-length Brpf1 (I-K; green) and the fragment containing the bromodomain and PWWP domain (L; green) co-localize with the active chromatin markers H2AK5Ac (I,L; red) and H3K4me1 (J; red), but not with the inactive chromatin marker H3K9me3 (K; red). Left panels are counterstained with DAPI (blue); merged images are shown in right-hand panels; regions with strong co-localization (yellow) are indicated by arrows. Scale bars: 2.5 µm in A; 5 µm in B,I-L.

View larger version (41K): [in this window] [in a new window]   Fig. 7. The bromodomain and PWWP domain bind histones. (A) Loading controls of GST-fused recombinant domains of Brpf1 (PHD finger, bromodomain, or PWWP domain) used in histone-binding assays (B-D). Relevant bands are indicated with asterisks. (B) Coomassie-staining of histones retained on glutathion beads without (A) or with (B) indicated GST-Brpf1 domains. Left lane shows 10% input of core histones used per assay. (C) Binding of purified H2A or H2B from calf serum with indicated GST-Brpf1 domains, analyzed by Coomassie staining. (D) Binding of core histones from untreated or butyrate-treated HeLa cells with indicated GST-Brpf1 domains, analyzed by western blotting with anti-H2AK5Ac (upper panel) or anti-H2A (lower panel) antibodies. The PWWP domain binds regular and hyperacetylated H2A equally well (compare lanes 5 and 6 of lower panel), whereas the bromodomain preferentially binds hyperacetylated H2A (compare lanes 3 and 4 of lower panel). This is also reflected in the higher relative signal intensity obtained with the anti-H2AK5Ac and the anti-pan H2A antibodies (compare upper and lower bands of lane 4 with those of lanes 6 and 8).

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